Simultaneous determination of tyrosine, phenylalanine and deoxyguanosine oxidation products by liquid chromatography–tandem mass spectrometry as non-invasive biomarkers for oxidative damage

Abstract

We developed an isotope dilution HPLC–atmospheric pressure chemical ionization–tandem mass spectrometry (HPLC–APCI–MS/MS) method for the simultaneous determination of p-tyrosine, phenylalanine, o, o′-dityrosine, m-tyrosine, o-tyrosine, 3-chlorotyrosine and 3-nitrotyrosine and 8-hydroxy-2′-deoxyguanosine (8-OHdG) that requires no extensive sample pre-treatment. p-[ 2H 4]Tyrosine and o, o′-[ 2H 6]dityrosine were used as internal standards. Calibration curves of the method were linear ( r 2=0.990–0.999) over a concentration range of 0.03–10 μM for o-tyrosine; 0.04–10 μM for 3-nitrotyrosine and 3-chlorotyrosine; 0.05–10 μM for o, o′-dityrosine; and for m-tyrosine; 1.0–100 μM for p-tyrosine and for phenylalanine; and 0.01–10 μM for 8-OHdG. The detection limits were from 0.025 to 0.05 μM for the tyrosine derivatives; 0.01 μM for 8-OHdG; and 0.5 μM for p-tyrosine and for phenylalanine, respectively. Within-day coefficients of variation (CV) for spiked human urine samples ranged from 2.7 to 7.0%, except for 8-OHdG (13.7%). Between-day variations ranged from 7.9 to 13.0%, except for o-tyrosine (CV = 18.2%), and for 8-OHdG (CV = 24.7%). The background levels of p-tyrosine, phenylalanine, o, o′-dityrosine, and o-tyrosine in morning urine of eight healthy volunteers were 3890±590, 3420±730, 5.8±0.3, and 9.2±1.5 μmol/mol creatinine, respectively. Using the present HPLC–APCI–MS/MS method, the urinary background levels of m-tyrosine, 3-chlorotyrosine, 3-nitrotyrosine and 8-OHdG were below the limit of detection.